Induced protein degradation for therapeutics: past, present, and future.

The concept of induced protein degradation by small molecules has emerged as a promising therapeutic strategy that is particularly effective in targeting proteins previously considered "undruggable." Thalidomide analogs, employed in the treatment of multiple myeloma, stand as prime examples. These compounds serve as molecular glues, redirecting the CRBN E3 ubiquitin ligase to degrade myeloma-dependency factors, IKZF1 and IKZF3. The clinical success of thalidomide analogs demonstrates the therapeutic potential of induced protein degradation. Beyond molecular glue degraders, several additional modalities to trigger protein degradation have been developed and are currently under clinical evaluation. These include heterobifunctional degraders, polymerization-induced degradation, ligand-dependent degradation of nuclear hormone receptors, disruption of protein interactions, and various other strategies. In this Review, we will provide a concise overview of various degradation modalities, their clinical applications, and potential future directions in the field of protein degradation.

TP63 fusions drive multicomplex enhancer rewiring, lymphomagenesis, and EZH2 dependence.

Gene fusions involving tumor protein p63 gene (TP63) occur in multiple T and B cell lymphomas and portend a dismal prognosis for patients. The function and mechanisms of TP63 fusions remain unclear, and there is no target therapy for patients with lymphoma harboring TP63 fusions. Here, we show that TP63 fusions act as bona fide oncogenes and are essential for fusion-positive lymphomas. Transgenic mice expressing TBL1XR1::TP63, the most common TP63 fusion, develop diverse lymphomas that recapitulate multiple human T and B cell lymphomas. Here, we identify that TP63 fusions coordinate the recruitment of two epigenetic modifying complexes, the nuclear receptor corepressor (NCoR)-histone deacetylase 3 (HDAC3) by the N-terminal TP63 fusion partner and the lysine methyltransferase 2D (KMT2D) by the C-terminal TP63 component, which are both required for fusion-dependent survival. TBL1XR1::TP63 localization at enhancers drives a unique cell state that involves up-regulation of MYC and the polycomb repressor complex 2 (PRC2) components EED and EZH2. Inhibiting EZH2 with the therapeutic agent valemetostat is highly effective at treating transgenic lymphoma murine models, xenografts, and patient-derived xenografts harboring TP63 fusions. One patient with TP63-rearranged lymphoma showed a rapid response to valemetostat treatment. In summary, TP63 fusions link partner components that, together, coordinate multiple epigenetic complexes, resulting in therapeutic vulnerability to EZH2 inhibition.

The human E3 ligase RNF185 is a regulator of the SARS-CoV-2 envelope protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks multiple human proteins during infection and viral replication. To examine whether any viral proteins employ human E3 ubiquitin ligases, we evaluated the stability of SARS-CoV-2 proteins with inhibition of the ubiquitin proteasome pathway. Using genetic screens to dissect the molecular machinery involved in the degradation of candidate viral proteins, we identified human E3 ligase RNF185 as a regulator of protein stability for the SARS-CoV-2 envelope protein. We found that RNF185 and the SARS-CoV-2 envelope co-localize to the endoplasmic reticulum (ER). Finally, we demonstrate that the depletion of RNF185 significantly increases SARS-CoV-2 viral titer in a cellular model. Modulation of this interaction could provide opportunities for novel antiviral therapies.

Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders.

Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics1-3. Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs4-6. The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans-labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). BRD4BD2, in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 Å cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4BD2 revealed that the loop conformation around BRD4His437, rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology.

Exploring the target scope of KEAP1 E3 ligase-based PROTACs.

Targeted protein degradation (TPD) uses small molecules to recruit E3 ubiquitin ligases into the proximity of proteins of interest, inducing ubiquitination-dependent degradation. A major bottleneck in the TPD field is the lack of accessible E3 ligase ligands for developing degraders. To expand the E3 ligase toolbox, we sought to convert the Kelch-like ECH-associated protein 1 (KEAP1) inhibitor KI696 into a recruitment handle for several targets. While we were able to generate KEAP1-recruiting degraders of BET family and murine focal adhesion kinase (FAK), we discovered that the target scope of KEAP1 was narrow, as targets easily degraded using a cereblon (CRBN)-recruiting degrader were refractory to KEAP1-mediated degradation. Linking the KEAP1-binding ligand to a CRBN-binding ligand resulted in a molecule that induced degradation of KEAP1 but not CRBN. In sum, we characterize tool compounds to explore KEAP1-mediated ubiquitination and delineate the challenges of exploiting new E3 ligases for generating bivalent degraders.

BTBBCL6 dimers as building blocks for reversible drug-induced protein oligomerization.

Here, we characterize the BTB domain of the transcription factor BCL6 (BTBBCL6) as a small-molecule-controlled, reversible oligomerization switch, which oligomerizes upon BI-3802 treatment and de-oligomerizes upon addition of BI-3812. We show that the magnitude of oligomerization can be controlled in vitro by BI-3802 concentration and exposure time. In cellular models, exposure to BI-3802/BI-3812 can drive multiple cycles of foci formation consisting of BTBBCL6 fused to EGFP, which are not degraded due to the lack of a degron. We generated an epidermal growth factor receptor (EGFR)-BTBBCL6 fusion. Treatment with BI-3802, as an ON switch, induced EGFR-BTBBCL6 phosphorylation and activation of downstream effectors, which could in part be reversed by the addition of BI-3812, as an OFF switch. Finally, BI-3802-induced oligomerization of the EGFR-BTBBCL6 fusion enhanced proliferation of an EGF-dependent cell line in absence of EGF. These results demonstrate the successful application of small-molecule-induced, reversible oligomerization as a switch for synthetic biology.

Identification of Thieno[3,2-d]pyrimidine Derivatives as Dual Inhibitors of Focal Adhesion Kinase and FMS-like Tyrosine Kinase 3.

Focal adhesion kinase (FAK) is overexpressed in highly invasive and metastatic cancers. To identify novel FAK inhibitors, we designed and synthesized various thieno[3,2-d]pyrimidine derivatives. An intensive structure-activity relationship (SAR) study led to the identification of 26 as a lead. Moreover, 26, a multitargeted kinase inhibitor, possesses excellent potencies against FLT3 mutants as well as FAK. Gratifyingly, 26 remarkably inhibits recalcitrant FLT3 mutants, including F691L, that cause drug resistance. Importantly, 26 is superior to PF-562271 in terms of apoptosis induction, anchorage-independent growth inhibition, and tumor burden reduction in the MDA-MB-231 xenograft mouse model. Also, 26 causes regression of tumor growth in the MV4-11 xenograft mouse model, indicating that it could be effective against acute myeloid leukemia (AML). Finally, in an orthotopic mouse model using MDA-MB-231, 26 remarkably prevents metastasis of orthotopic tumors to lymph nodes. Taken together, the results indicate that 26 possesses potential therapeutic value against highly invasive cancers and relapsed AML.

Small-molecule-induced polymerization triggers degradation of BCL6.

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets1-3, but a substantial subset of proteins are resistant to targeted degradation using existing approaches4,5. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-à-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL66. We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.

Rationally Designed Covalent BCL6 Inhibitor That Targets a Tyrosine Residue in the Homodimer Interface.

B-cell lymphoma 6 (BCL6) is a transcriptional repressor frequently deregulated in lymphoid malignancies. BCL6 engages with number of corepressors, and these protein-protein interactions are being explored as a strategy for drug development. Here, we report the development of an irreversible BCL6 inhibitor TMX-2164 that uses a sulfonyl fluoride to covalently react with the hydroxyl group of Tyrosine 58 located in the lateral groove. TMX-2164 exhibits significantly improved inhibitory activity compared to that of its reversible parental compound and displays sustained target engagement and antiproliferative activity in cells. TMX-2164 therefore represents an example of a tyrosine-directed covalent inhibitor of BCL6 which demonstrates advantages relative to reversible targeting.

Targeted protein degradation as a powerful research tool in basic biology and drug target discovery.

Controlled perturbation of protein activity is essential to study protein function in cells and living organisms. Small molecules that hijack the cellular protein ubiquitination machinery to selectively degrade proteins of interest, so-called degraders, have recently emerged as alternatives to selective chemical inhibitors, both as therapeutic modalities and as powerful research tools. These systems offer unprecedented temporal and spatial control over protein function. Here, we review recent developments in this field, with a particular focus on the use of degraders as research tools to interrogate complex biological problems.

Structural complementarity facilitates E7820-mediated degradation of RBM39 by DCAF15.

The investigational drugs E7820, indisulam and tasisulam (aryl-sulfonamides) promote the degradation of the splicing factor RBM39 in a proteasome-dependent mechanism. While the activity critically depends on the cullin RING ligase substrate receptor DCAF15, the molecular details remain elusive. Here we present the cryo-EM structure of the DDB1-DCAF15-DDA1 core ligase complex bound to RBM39 and E7820 at a resolution of 4.4 Å, together with crystal structures of engineered subcomplexes. We show that DCAF15 adopts a new fold stabilized by DDA1, and that extensive protein-protein contacts between the ligase and substrate mitigate low affinity interactions between aryl-sulfonamides and DCAF15. Our data demonstrate how aryl-sulfonamides neo-functionalize a shallow, non-conserved pocket on DCAF15 to selectively bind and degrade RBM39 and the closely related splicing factor RBM23 without the requirement for a high-affinity ligand, which has broad implications for the de novo discovery of molecular glue degraders.

Identification of Novel Resorcinol Amide Derivatives as Potent and Specific Pyruvate Dehydrogenase Kinase (PDHK) Inhibitors.

Pyruvate dehydrogenase kinases (PDHKs) promote abnormal respiration in cancer cells. Studies with novel resorcinol amide derivatives based on VER-246608 (6) led to the identification of 19n and 19t containing five-membered heteroaromatic rings as unique structural features. These substances possess single-digit nanomolar activities against PDHKs. 19t exhibits higher potencies against PDHK1/2/4 than does 6 and inhibits only PDHKs among 366 kinases. Moreover, 19g, 19l, and 19s were found to be isotype-selective PDHK inhibitors. Molecular dynamics simulations provide a better understanding of how the heteroaromatic rings affect the activities of 19n and 19t on PDHK1/2/3/4. Moreover, 19n possesses a much higher antiproliferative activity against cancer cells than does 6. We demonstrated that the results of PDH assays better correlate with cellular activities than do those of PDHK kinase assays. Furthermore, 19n induces apoptosis of cancer cells via mitochondrial dysfunction, suppresses tumorigenesis, and displays a synergistic effect on satraplatin suppression of cancer cell proliferation.

Asymmetric Synthesis of (-)-6-Desmethyl-Fluvirucinine A1 via Conformationally-Controlled Diastereoselective Lactam-Ring Expansions.

The versatile synthesis of (-)-6-desmethyl-fluvirucinine A1 was accomplished at a 24% overall yield through a thirteen-step process from a known vinylpiperidine. The key part involved the elaboration of the distal stereocenters and a macrolactam skeleton via conformationally-induced diastereocontrol and the iterative aza-Claisen rearrangements of lactam precursors.

In situ imaging of quantum dot-AZD4547 conjugates for tracking the dynamic behavior of fibroblast growth factor receptor 3.

Fibroblast growth factor receptors (FGFRs) play an important role in determining cell proliferation, differentiation, migration, and survival. Although a variety of small-molecule FGFR inhibitors have been developed for cancer therapeutics, the interaction between FGFRs and FGFR inhibitors has not been well characterized. The FGFR-inhibitor interaction can be characterized using a new imaging probe that has strong, stable signal properties for in situ cellular imaging of the interaction without quenching. We developed a kinase-inhibitor-modified quantum dot (QD) probe to investigate the interaction between FGFR and potential inhibitors. Especially, turbo-green fluorescent protein-FGFR3s were overexpressed in HeLa cells to investigate the colocalization of FGFR3 and AZD4547 using the QD-AZD4547 probe. The result indicates that this probe is useful for investigating the binding behaviors of FGFR3 with the FGFR inhibitor. Thus, this new inhibitor-modified QD probe is a promising tool for understanding the interaction between FGFR and inhibitors and for creating future high-content, cell-based drug screening strategies.

Identification of a novel 5-amino-3-(5-cyclopropylisoxazol-3-yl)-1-isopropyl-1H-pyrazole-4-carboxamide as a specific RET kinase inhibitor.

Activating mutations of REarrange during Transfection (RET) kinase frequently occur in human thyroid and lung cancers. An enormous effort has been devoted to discover potent and selective inhibitors of RET. Selective and potent inhibitors against constitutively active RET mutants are rare to date as identification of selective RET inhibitors is challenging. In a recent effort we identified a novel and specific RET inhibitor of 5-aminopyrazole-4-carboxamide scaffold, which was designed to enhance the metabolic stability of the pyrazolopyrimidine scaffold. In the SAR study described in the current report, we identified the 5-aminopyrazole-4-carboxamide analog 15l, which displays high metabolic stability. Compound 15l is potent against gatekeeper mutant (IC50 = 252 nM) of RET as well as against wild-type RET (IC50 = 44 nM). This substance effectively suppresses growth of Ba/F3 cells transformed with wild-type RET and its gatekeeper mutant (V804M), and thyroid-cancer derived TT cells while it does not affect parental Ba/F3 cells and Nthy ori-3-1, normal thyroid cells. Also, the results of a global kinase profiling assay on a panel of 369 kinases, show that 15l exclusively inhibits RET. Based on its exceptional kinase selectivity, great potency and metabolic stability, 15l represents a promising lead for the discovery of RET directed therapeutic agent and should be a key tool in studies aimed at understanding RET biology.

Double Blockade of Glioma Cell Proliferation and Migration by Temozolomide Conjugated with NPPB, a Chloride Channel Blocker.

Glioblastoma is the most common and aggressive primary malignant brain tumor. Temozolomide (TMZ), a chemotherapeutic agent combined with radiation therapy, is used as a standard treatment. The infiltrative nature of glioblastoma, however, interrupts effective treatment with TMZ and increases the tendency to relapse. Voltage-gated chloride channels have been identified as crucial regulators of glioma cell migration and invasion by mediating cell shape and volume change. Accordingly, chloride current inhibition by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), a chloride channel blocker, suppresses cell movement by diminishing the osmotic cell volume regulation. In this study, we developed a novel compound, TMZ conjugated with NPPB (TMZ-NPPB), as a potential anticancer drug. TMZ-NPPB blocked chloride currents in U373MG, a severely invasive human glioma cell line, and suppressed migration and invasion of U373MG cells. Moreover, TMZ-NPPB exhibited DNA modification activity similar to that of TMZ, and surprisingly showed remarkably enhanced cytotoxicity relative to TMZ by inducing apoptotic cell death via DNA damage. These findings indicate that TMZ-NPPB has a dual function in blocking both proliferation and migration of human glioma cells, thereby suggesting its potential to overcome challenges in current glioblastoma therapy.

A Pyrazolo[3,4-d]pyrimidin-4-amine Derivative Containing an Isoxazole Moiety Is a Selective and Potent Inhibitor of RET Gatekeeper Mutants.

Aberrant RET kinase signaling plays critical roles in several human cancers such as thyroid carcinoma. The gatekeeper mutants (V804L or V804M) of RET are resistant to currently approved RET inhibitors such as cabozantinib and vandetanib. We, for the first time, report a highly selective and extremely potent RET inhibitor, 6i rationally designed. Compound 6i inhibits strongly RET gatekeeper mutants and other clinically relevant RET mutants as well as wt-RET. This substance also significantly suppresses growth of thyroid cancer-derived TT cell lines and Ba/F3 cells transformed with various RET mutants. Docking studies reveal that the isoxazole moiety in 6i is responsible for binding affinity improvement by providing additional site for H-bonding with Lys758. Also, 6i not only substantially blocks cellular RET autophosphorylation and its downstream pathway, it markedly induces apoptosis and anchorage-independent growth inhibition in TT cell lines while having no effect on normal thyroid Nthy ori-3-1 cells.

Identification of novel therapeutic targets in acute leukemias with NRAS mutations using a pharmacologic approach.

Oncogenic forms of NRAS are frequently associated with hematologic malignancies and other cancers, making them important therapeutic targets. Inhibition of individual downstream effector molecules (eg, RAF kinase) have been complicated by the rapid development of resistance or activation of bypass pathways. For the purpose of identifying novel targets in NRAS-transformed cells, we performed a chemical screen using mutant NRAS transformed Ba/F3 cells to identify compounds with selective cytotoxicity. One of the compounds identified, GNF-7, potently and selectively inhibited NRAS-dependent cells in preclinical models of acute myelogenous leukemia and acute lymphoblastic leukemia. Mechanistic analysis revealed that its effects were mediated in part through combined inhibition of ACK1/AKT and of mitogen-activated protein kinase kinase kinase kinase 2 (germinal center kinase). Similar to genetic synthetic lethal approaches, these results suggest that small molecule screens can be used to identity novel therapeutic targets in cells addicted to RAS oncogenes.